In optical microscopy, particularly in high resolution optical microscopy, an immersion fluid with a specific index of refraction is used in the working space between an objective lens and a sample slide. Using an immersion fluid with an index of refraction that is greater than air's index of refraction and closer to the objective lens's index of refraction enables the collection of more light with the objective lens, and results in improved resolution for the optical microscope.
When using an immersion fluid such as immersion fluid, it is imperative that the objective lens and the sample slide are both in direct contact with the immersion fluid. The presence of an air gap between the sample slide and the objective lens significantly decreases the advantage of using an immersion fluid. While it is critical to position both the objective lens and sample slide close enough to each other so that both are in contact with the immersion fluid, it is equally important to keep the objective lens from directly contacting the sample slide. Direct contact between the sample slide and the objective lens may result in damage to one or both of the sample and the objective lens. The objective lens is a key component of an optical microscope, and it is expensive to replace. Additionally, samples may be difficult, expensive, and time-consuming to prepare for imaging.
During a typical workflow, the distance between the objective lens and the sample slide is adjusted to bring the optical microscope into focus. During the process of focusing the optical microscope, it is often difficult for the operator to accurately determine whether the objective lens and the sample slide are both in direct contact with the immersion fluid. As discussed above, it is desirable to eliminate the air gap between the sample slide and the objective lens yet avoid causing the objective lens to make direct contact with the sample slide. The exact thickness of the sample is highly variable on the scale of many micrometers. As such, it is not possible to accurately predict the focus height of the objective lens with respect to each unique sample.
For these and other reasons, an improved optical microscope and method of determining lens immersion in optical microscopy is desired.